Knowledge of the nature of a reaction's transition state is required for understanding of enzymatic catalysis, as catalysis is defined as stabilization of a transition state relative to reactants. Phosphoryl transfer, typically involving high energy phosphate donors such as ATP constitutes the most common class of biological reactions. Despite the importance of this reaction, the transition state for phosphoryl transfer from ATP in solution has not been established. We are therefore studying these solution reactions using approaches of physical organic chemistry in order to provide a basis for understanding enzymatic phosphoryl transfer. The transfer of phosphate from ATP to a series of alcohols is under investigation; these reactions are analogous to phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. A very small dependence of the rate of transfer on the nucleophilicity (pKa) of the alcohol is observed. This implies that there is a small amoung of bond formation to the incoming alcohol nucleophile and is consistent with a dissociative, metaphosphate-like transition state. In addition, coordination of Mg2+ to ATP has no significant effect on the relative reactivity of the alcohols, suggesting that Mg2+ does not alter the nature of this dissociative transition state. This is in contrast to several literature proposals. To complete the characterization of the transition state for phosphoryl transfer from ATP, we are studying the dependence of the rate of transfer on the leaving group of the reaction. To that end we have synthesized ATP analogs of varying leaving group stability to determine the extent of bond breakage to the leaving group in the transition state. By assisting in identification of the ATP analogs -- a series of pyrophosphonates -- mass spectrometry performed at the UCSF Facility will enable valid interpretation of kinetic data. Recent results indicate that the transition state remains dissociate for phosphoryl transfer catalyzed by E. coli alkaline phosphatase (F. Hollfelder & D.H., in preparation). These findings suggest that some enzymes have developed strategies for stabilizing a dissociative transition state for phosphoryl transfer; it will be interesting to discover whether or not other enzymes instead alter the nature of the transition state.